Regulatory agencies throughout the world routinely require data on the toxicity of new drugs as a part of the safely evaluation process. The primary objective of the safety evaluation process is to collect information that is indicative of whether the benefits of a using a potential new pharmaceutical compound (e.g., test compound) as a therapeutic agent outweighs the risks and side effects associated with its use. Today, it is not possible to register a new drug without providing information regarding its genotoxic (i.e., mutagenic or carcinogenic) potential.
Although no specific tests are mandated, there is a consensus that recommends the use of a standard four-test battery including: a gene mutation assay in bacteria; an in vitro test for chromosome abberations in mammalian cells; an in vitro test for gene mutation in eukaryotic cells; and an in vivo test for genetic damage. Based on the observation of Dr. Bruce Ames and his colleagues that most carcinogens are also mutagens, numerous test systems (e.g., prokaryotic and eukaryotic) have been developed over the past several years for the purpose of evaluating the genotoxic activity of pharmaceutical compounds and/or their reactive metabolites in various in vitro assays. More specifically, determination of the mutagenic potential of test compounds have historically involved the use of microbial mutation assays, particularly Salmonella reversion assays (Ames, B. N., et al., (1975) Mutation Res. 31:347-363).
While it is recognized that a drug or new chemical entity can be toxic at different levels, drug-induced mutagenesis of DNA (genotoxicity) underlies many decisions to stop the development of candidate drugs. Generally speaking, genotoxicity can take the form of gene mutation, structural chromosomal abberations, recombination and numerical changes. The standard Ames Assay, which is a cornerstone in the field of toxicology, utilizes several different tester strains, each with a distinct mutation (e.g., transition, frameshift etc.) in one of the genes comprising the histidine (his) biosynthetic operon (Ames, B. N., et al., (1975) Mutation Res. 31:347-363). The detection of revertant (i.e., mutant) bacteria in test samples that are capable of growth in the absence of histidine indicates that the compound under evaluation is characterized by genotoxic (i.e. mutagenic) activity. The Ames Assay is capable of detecting several different types of mutations (genetic damage) which may occur in one or more of the tester strains. As mentioned above, the practice of using an in vitro bacterial assay to evaluate the genotoxic activity of drug candidates is based on the prediction that a substance that is mutagenic in a bacterium is likely to be carcinogenic in laboratory animals, and by extension may be carcinogenic or mutagenic to humans. An extensive database containing the results of toxicity data obtained in a traditional bacterial reverse mutation test has been established (McCann, J., et al., (1975) Proc. Natl. Acad. Sci. USA 72:5135-9). Generally speaking, the Ames Assay detects the genotoxic activity of carcinogenic/mutagenic compounds belonging to diverse chemical classes with an efficiency of about 80%.
In practice, the Ames Assay is relatively cumbersome to perform because multiple tester strains are necessary due to the fact that chemical mutagens are specific to the type of DNA alteration that they can affect. The standard plate assay utilizes multiple 100-mm dishes and consumes a relatively large amount of compound (i.e., hundreds of milligrams to gram quantities) and can cost from $4000-$5000 per sample.
The requirement for relatively large amounts of compound is also attributed to the fact that in order to increase the probability of identifying DNA-reactive (i.e., genotoxic) compounds, the toxicity of each compound being evaluated is typically tested at several doses on multiple genetically distinct tester strains. Thus, it is understandable that under the traditional paradigm of drug discovery, the Ames reversion test is routinely performed relatively late in the drug discovery process.
Recent advances in the fields of combinatorial chemistry and high throughput screening has brought the drug discovery process to a point where large numbers of molecules with great diversity can be readily synthesized and evaluated for biological activity. The incorporation of combinatorial chemistry into the drug development process has left some companies with a backlog of hundreds of thousands of compounds, many of which may be available in limited quantities, waiting to be tested as therapeutic agents. Because the financial investment in drug discovery increases exponentially as a compound progresses from initial discovery, through development and registration, there is a heightened need for more efficient means of evaluating the safety of new chemical entities at a relatively early stage of the drug development process.
The development of a high throughput genotoxicity assay could potentially save significant amounts of time and money by allowing investigators to eliminate compounds with genotoxic activity at an early stage of the drug development process. Thus, the development of a sensitive, bacterial genotoxic assay with by low compound requirements, that is amenable to automation, and which facilitates high throughput screening formats addresses an unmet need in the field of genetic toxicology.